DNA is an underrepresented target for small molecule therapeutic agents. One reason for the dearth of DNA-targeted drugs is that the fundamental molecular mechanisms that govern sequence and structural-selective ligand binding to DNA are poorly understood. The rational design of new DNA-targeted drugs requires a thorough understanding of the binding mechanisms of existing compounds that bind to DNA with unique types of specificity. The long-range goal of the proposed research is to understand the molecular mechanism of intercalation reactions, with emphasis on the energetic basis of their sequence and structural selective binding. Renewal is sought for a successful and highly productive basic science program that has produced several promising avenues for DNA-targeted drug development. Research in the next funding period will focus on innovative studies designed to clarify the mechanisms that govern structural and sequence selective ligand binding to nucleic acids. Specific aims include: 1. Use and refinement of a highthroughput competition dialysis assay for the identification and characterization of structural selective ligand binding to nucleic acids. 2. Characterization of ligand binding to DNA:RNA hybrid structures. 3. Characterization of regional-selective drug binding by a novel calorimetric "bootprinting" method. The results of these studies will: 1. Provide a definitive taxonomy of structural selective ligand binding to nucleic acids, along with a description of the energetic basis of that structural selectivity, 2. Provide fundamental information to guide the rational design of compounds targeted to DNA:RNA hybrid structures, a target of enormous biological significance. 3. Provide a new tool for the quantitative study of drug-DNA interaction in long (genomic) pieces of DNA.